The signaling molecule Sonic Hedgehog (SHH) plays a critical role in patterning the ventral midbrain of vertebrates. Our recent studies have established that the requirement for Hedgehog (HH) signaling in the chick midbrain is modulated spatially and temporally in a complex manner across the midbrain anlage. Unfortunately, the patterns of expression of downstream regulators that might modulate the HH signal in the midbrain are not currently known. To fill this gap, we have examined across time, the expression pattern of 14 genes that function in the HH signaling cascade in the midbrain and spinal cord. Our results suggest that SHH expression in the axial mesendoderm begins before the expression of known HH receptors/HH-binding proteins (e.g., PTC1, PTC2, HHIP, BOC, MEGALIN). In the midbrain, PTC and GLI genes are expressed and then eliminated very early from the ventral midline. However, they exhibit high and persistent expression in the midbrain region circumscribing the SHH source. Intriguingly, multiple HH-binding proteins (BOC, MEGALIN) and HH effectors (GLI1-3, SMO, SUFU, DZIP) are expressed in the dorsal midbrain and the midbrain–hindbrain boundary. Finally, we report for the first time that IHH is expressed in intermediate regions of the spinal cord, where its expression does not overlap with that of SHH. Developmental Dynamics 236:1363–1373, 2007. © 2007 Wiley-Liss, Inc.